An important challenge in cell biology is to describe the force-generating mechanisms for such essential eukaryotic processes as exocytosis, endocytosis, and overall cell motility. In recent years, a single-headed, nonfilamentous form of myosin, called myosin I, has emerged as a likely motor to support actin-dependent membrane motility. Until now, myosin I had been purified and partially characterized only from protozoans and avian intestinal brush border. We have now succeeded in isolating mammalian forms of the enzyme from bovine adrenal medulla, adrenal cortex, and brain. This proposal focuses on two major questions regarding mammalian myosin I. The first is the regulation of the enzyme's catalytic activity and its interaction with actin and membranes and the second is its role in neuroendocrine cell function. To study the regulation of myosin I we will clone it and identify and characterize domains on the molecule which interact with F-actin, membranes, and calmodulin. The role of Ca2+ and myosin I phosphorylation in regulating these functional interactions will be investigated, both in vitro and in cultured adrenal chromaffin cells. To address the question of the physiological function of myosin I, we will first localize the protein in resting and stimulated chromaffin cells and in PC12 cells before and after they have been induced to differentiate into neuron-like cells by nerve growth factor. Subcellular fractionation will reveal whether myosin I is selectively targeted to specific organelles. If so, the basis for this selectivity will be investigated. Finally, we will introduce antibodies into cultured chromaffin cells. The effect of these antibodies on the exocytosis/endocytosis cycle and on catecholamine release will be examined. This proposal will provide a structural and functional characterization of the mammalian myosin I enzyme and will lay the foundation for future studies on the role of myosin I as an intracellular motor.